Junctophilin-2表达下调介导心肌细胞兴奋—收缩耦联结构与功能的重塑

目的阐明横管(T-tubule,TT)与肌质网(sarcoplasmic reticulum,SR)耦联关键蛋白junctophilin-2(JP2)的表达下调对心肌细胞二联体超微结构以及兴奋-收缩耦联功能的影响。方法通过构建JP2特异性shRNA腺病毒敲减成年大鼠心肌细胞中JP2的表达后,采用透射电子显微镜观测心肌细胞横管和肌质网二联体结构的形态变化,并进一步采用全细胞膜片钳结合激光共聚焦钙成像技术检测心肌细胞兴奋收缩联联功能的变化。结果通过腺病毒敲减心肌细胞JP2表达后,心肌细胞内总TT数目、TT与SR耦联的二联体结构数目以及单个耦联子内耦联SR的TT长度占TT周长的百分比均显著降低;与此同时,JP2表达下降后心肌细胞钙瞬变幅度降低,收缩功能减弱,表现出与心力衰竭类似的表型。结论心肌细胞JP2表达下降引起心肌细胞兴奋-收缩耦联结构与功能的损伤,为心力衰竭病理情况下心肌细胞结构与功能的调控机制提供了直接有力的实验证据。

Structural and functional remodeling of excitation-contraction coupling mediated by junctophilin-2 down-regulation in cardiomyocytes

Objective To investigate whether down-regulation of junctophilin-2 （JP2） induced the impairment of dyad structure （T-tubule and sarcoplasmic reticulum） and excitation-contraction coupling （E-C coupling） function in adult rat cardiomyocytes. Methods We delivered JP-specific shRNA into adult rat cardiomyocytes by adenovirus to knock down JP2 expression. The morphology of T-tubules （TTs） and sarcoplasmic reticulum （SR）, and the dyad structure were examined by transmission electronic microscopy. Whole cell patch clamp combined with confocal Ca2＋ imaging technique was applied to record L-type Ca2＋ current and Ca2＋ transient in cardiomyocytes. Results The expression level of JP2 in adult cardiomyocytes was successfully suppressed by JP2-specific shRNA. The number of total TTs and TTs apparently coupled with SRs were greatly reduced by JP2 down-regulation. Coupled rate, calculated from the ratio of TT length apparently coupled with SR and TT perimeter, was also significantly decreased in JP2 shRNA group. Moreover, JP2 down-regulation weakened the amplitude of Ca2＋ transient and the efficiency of E-C coupling, similarly to failing heart cells. Conclusion The ultrastructure and function of E-C coupling were impaired by down-regulation of JP2, which provides the direct evidence of mechanism underlying the structural and functional regulation of cardiomyocyte in heart failure.